Purification and partial characterization of English sole (Pleuronectes vetulus) vitellogenin.
Vitellogenin (VTG) was purified by double-step chromatography from plasma of male English sole treated with 17 beta-estradiol. The intact protein appeared to exist as a dimer in two forms of approximately 300 and 320 kDa and had an isoelectric point of 6.63. In SDS-PAGE, it was reduced to a single monomer of approximately 130 kDa. In immunoblotting, the protein showed cross-reactivity with coho salmon VTG antiserum. Native PAGE (sample not treated with the reducing agent mercaptoethanol) and immunoblotting of plasma from control and estradiol-treated male sole and gravid female sole demonstrated that the putative English sole VTG was normally female specific and estradiol inducible in males. It was immunocytochemically localized in liver and ovary of English sole, rock sole and starry flounder, using polyclonal antiserum to the purified protein from the estradiol-treated male English sole. The protein was characterized as a phospholipoglycoprotein by native PAGE, staining the gels for phosphorus with methyl green, for lipid with Sudan black B and for carbohydrate by an improved periodic-acid Fuchsin sulfite method. The amino acid composition of the putative VTG was generally similar to that of VTGs from other teleosts, with the non-polar amino acids alanine, valine, leucine and isoleucine accounting for one-third of the total amino acids present. However, English sole vitellogenin contained a higher proportion of leucine and a lower proportion of glycine than most other teleost vitellogenins isolated to date.
Roubal
W T
WT
Environmental Conservation Division, Northwest Fisheries Science Center, NMFS, Seattle, WA 98112, USA.
Lomax
D P
DP
Willis
M L
ML
Johnson
L L
LL
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D005412:Flatfishes; D007150:Immunohistochemistry; D008099:Liver / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D010053:Ovary / Q000378:metabolism; D012474:Salmon; D014819:Vitellogenins / Q000097:blood / Q000276:immunology* / Q000302:isolation & purification*
D000596:Amino Acids; D014819:Vitellogenins; D004958:Estradiol